Endocannabinoids (eCBs) mediate one of the most prevalent and complex signaling systems in the central nervous system. They are ubiquitous regulators of excitatory and inhibitory neural transmission, and studies over the past two decades have well established a retrograde mechanism of eCB signaling. Specifically, postsynaptic neurons release eCBs, which activate presynaptically located eCB receptor type 1 (CB1R) and regulate release of neurotransmitters. This retrograde mechanism has become the central tenet of theories describing how eCBs regulate synaptic transmission, which they do at both excitatory and inhibitory synapses in many brain regions. However, we have recently acquired data from dopaminergic projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) that depict a scenario in distinct contrast to this dominant concept. Using optogenetic, electron microscopic and electrophysiological tools, our preliminary studies demonstrate a form of short-term synaptic plasticity that is likely triggered by eCBs released from dopaminergic axon terminals. Briefly, i) Selective activation of VTA-to-NAc dopaminergic axons triggers short- term inhibition of GABA transmission at adjacent synapses; ii) Activation of GABAergic synapses alone does not trigger this effect; iii) Inhibiting CB1Rs prevents this heterosynaptic plasticity; iv) The plasticity persists following postsynaptic chelation of Ca2+ by BAPTA, and in the presence of inotropic and metabotropic glutamate receptor blockers; v) Anandamide (AEA) is one of the two major eCBs in the CNS, and N- acylphosphatidylethanolamine phospholipase D (NAPE-PLD), an enzyme that synthesizes AEA, is identified in presynaptic terminals within the NAc. These results raise a striking possibility tat eCBs may be synthesized and released from dopaminergic presynaptic terminals and regulate adjacent GABAergic synapses in a cross- synaptic manner. This novel form of eCB-mediated cellular behavior, if verified, represents the first demonstration of presynaptic release of eCBs i the NAc. This CEBRA R21 application will more thoroughly characterize this exciting new form of eCB release. We will achieve this goal by thoroughly characterizing eCB release from VTA-to-NAc dopaminergic projections with definitive approaches, including optogenetic, pharmacological, molecular, and electron microscopic assays. Specifically, we will determine the type(s) of eCB that mediates the heterosynaptic regulation, the synthetic enzyme(s) that produces the eCB, and the pre- or postsynaptic sites where the eCB is released. The expected results will clarify whether presynaptic dopaminergic terminals do indeed release eCBs within the NAc, and thus potentially provide a new direction for understanding the cellular and behavioral roles of the eCB system. Given that eCBs are implicated in the pathophysiology of several brain diseases, including drug addiction, depression, and obesity, this line of research is highly relevant to the mission of the NIH.